Deletion of myeloid HDAC3 promotes efferocytosis to ameliorate retinal ischemic injury.

Ischemia-induced retinopathy is a hallmark finding of common visual disorders including diabetic retinopathy (DR) and central retinal artery and vein occlusions. Treatments for ischemic retinopathies fail to improve clinical outcomes and the design of new therapies will depend on understanding the underlying disease mechanisms. Histone deacetylases (HDACs) are an enzyme class that removes acetyl groups from histone and non-histone proteins, thereby regulating gene expression and protein function. HDACs have been implicated in retinal neurovascular injury in preclinical studies in which nonspecific HDAC inhibitors mitigated retinal injury. Histone deacetylase 3 (HDAC3) is a class I histone deacetylase isoform that plays a central role in the macrophage inflammatory response. We recently reported that myeloid cells upregulate HDAC3 in a mouse model of retinal ischemia-reperfusion (IR) injury. However, whether this cellular event is an essential contributor to retinal IR injury is unknown. In this study, we explored the role of myeloid HDAC3 in ischemia-induced retinal neurovascular injury by subjecting myeloid-specific HDAC3 knockout (M-HDAC3 KO) and floxed control mice to retinal IR. The M-HDAC3 KO mice were protected from retinal IR injury as shown by the preservation of inner retinal neurons, vascular integrity, and retinal thickness. Electroretinography confirmed that this neurovascular protection translated to improved retinal function. The retinas of M-HDAC3 KO mice also showed less proliferation and infiltration of myeloid cells after injury. Interestingly, myeloid cells lacking HDAC3 more avidly engulfed apoptotic cells in vitro and after retinal IR injury in vivo compared to wild-type myeloid cells, suggesting that HDAC3 hinders the reparative phagocytosis of dead cells, a process known as efferocytosis. Further mechanistic studies indicated that although HDAC3 KO macrophages upregulate the reparative enzyme arginase 1 (A1) that enhances efferocytosis, the inhibitory effect of HDAC3 on efferocytosis is not solely dependent on A1. Finally, treatment of wild-type mice with the HDAC3 inhibitor RGFP966 ameliorated the retinal neurodegeneration and thinning caused by IR injury. Collectively, our data show that HDAC3 deletion enhances macrophage-mediated efferocytosis and protects against retinal IR injury, suggesting that inhibiting myeloid HDAC3 holds promise as a novel therapeutic strategy for preserving retinal integrity after ischemic insult.

Effects of confounding and effect-modifying lifestyle, environmental and medical factors on risk of radiation-associated cardiovascular disease.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide. It has been known for some considerable time that radiation is associated with excess risk of CVD. A recent systematic review of radiation and CVD highlighted substantial inter-study heterogeneity in effect, possibly a result of confounding or modifications of radiation effect by non-radiation factors, in particular by the major lifestyle/environmental/medical risk factors and latent period. METHODS: We assessed effects of confounding by lifestyle/environmental/medical risk factors on radiation-associated CVD and investigated evidence for modifying effects of these variables on CVD radiation dose-response, using data assembled for a recent systematic review. RESULTS: There are 43 epidemiologic studies which are informative on effects of adjustment for confounding or risk modifying factors on radiation-associated CVD. Of these 22 were studies of groups exposed to substantial doses of medical radiation for therapy or diagnosis. The remaining 21 studies were of groups exposed at much lower levels of dose and/or dose rate. Only four studies suggest substantial effects of adjustment for lifestyle/environmental/medical risk factors on radiation risk of CVD; however, there were also substantial uncertainties in the estimates in all of these studies. There are fewer suggestions of effects that modify the radiation dose response; only two studies, both at lower levels of dose, report the most serious level of modifying effect. CONCLUSIONS: There are still large uncertainties about confounding factors or lifestyle/environmental/medical variables that may influence radiation-associated CVD, although indications are that there are not many studies in which there are substantial confounding effects of these risk factors.

A Review of Numerical Models of Radiation Injury and Repair Considering Subcellular Targets and the Extracellular Microenvironment.

Astronauts in space are subject to continuous exposure to ionizing radiation. There is concern about the acute and late-occurring adverse health effects that astronauts could incur following a protracted exposure to the space radiation environment. Therefore, it is vital to consider the current tools and models used to describe and study the organic consequences of ionizing radiation exposure. It is equally important to see where these models could be improved. Historically, radiobiological models focused on how radiation damages nuclear deoxyribonucleic acid (DNA) and the role DNA repair mechanisms play in resulting biological effects, building on the hypotheses of Crowther and Lea from the 1940s and 1960s, and they neglected other subcellular targets outside of nuclear DNA. The development of these models and the current state of knowledge about radiation effects impacting astronauts in orbit, as well as how the radiation environment and cellular microenvironment are incorporated into these radiobiological models, aid our understanding of the influence space travel may have on astronaut health. It is vital to consider the current tools and models used to describe the organic consequences of ionizing radiation exposure and identify where they can be further improved.

The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis.

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein µ1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was nonlethal in wild-type mice, whereas more than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, as well as extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 than T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a nonlethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCE Reovirus is a nonenveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the nonlethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.

An optimized method for the isolation of urinary extracellular vesicles for molecular phenotyping: detection of biomarkers for radiation exposure.

BACKGROUND: Urinary extracellular vesicles (EVs) are a source of biomarkers with broad potential applications across clinical research, including monitoring radiation exposure. A key limitation to their implementation is minimal standardization in EV isolation and analytical methods. Further, most urinary EV isolation protocols necessitate large volumes of sample. This study aimed to compare and optimize isolation and analytical methods for EVs from small volumes of urine. METHODS: 3 EV isolation methods were compared: ultracentrifugation, magnetic bead-based, and size-exclusion chromatography from 0.5 mL or 1 mL of rat and human urine. EV yield and mass spectrometry signals (Q-ToF and Triple Quad) were evaluated from each method. Metabolomic profiling was performed on EVs isolated from the urine of rats exposed to ionizing radiation 1-, 14-, 30- or 90-days post-exposure, and human urine from patients receiving thoracic radiotherapy for the treatment of lung cancer pre- and post-treatment. RESULTS: Size-exclusion chromatography is the preferred method for EV isolation from 0.5 mL of urine. Mass spectrometry-based metabolomic analyses of EV cargo identified biochemical changes induced by radiation, including altered nucleotide, folate, and lipid metabolism. We have provided standard operating procedures for implementation of these methods in other laboratories. CONCLUSIONS: We demonstrate that EVs can be isolated from small volumes of urine and analytically investigated for their biochemical contents to detect radiation induced metabolomic changes. These findings lay a groundwork for future development of methods to monitor response to radiotherapy and can be extended to an array of molecular phenotyping studies aimed at characterizing EV cargo.

Spaceflight induces oxidative damage to blood-brain barrier integrity in a mouse model.

Many factors contribute to the health risks encountered by astronauts on missions outside Earth's atmosphere. Spaceflight-induced potential adverse neurovascular damage and late neurodegeneration are a chief concern. The goal of the present study was to characterize the effects of spaceflight on oxidative damage in the mouse brain and its impact on blood-brain barrier (BBB) integrity. Ten-week-old male C57BL/6 mice were launched to the International Space Station (ISS) for 35 days as part of Space-X 12 mission. Ground control (GC) mice were maintained on Earth in flight hardware cages. Within 38 ± 4 hours after returning from the ISS, mice were euthanized and brain tissues were collected for analysis. Quantitative assessment of brain tissue demonstrated that spaceflight caused an up to 2.2-fold increase in apoptosis in the hippocampus compared to the control group. Immunohistochemical analysis of the mouse brain revealed an increased expression of aquaporin4 (AQP4) in the flight hippocampus compared to the controls. There was also a significant increase in the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) and a decrease in the expression of the BBB-related tight junction protein, Zonula occludens-1 (ZO-1). These results indicate a disturbance of BBB integrity. Quantitative proteomic analysis showed significant alterations in pathways responsible for neurovascular integrity, mitochondrial function, neuronal structure, protein/organelle transport, and metabolism in the brain after spaceflight. Changes in pathways associated with adhesion and molecular remodeling were also documented. These data indicate that long-term spaceflight may have pathological and functional consequences associated with neurovascular damage and late neurodegeneration.

Late Health Effects of Partial Body Irradiation Injury in a Minipig Model Are Associated with Changes in Systemic and Cardiac IGF-1 Signaling.

Clinical, epidemiological, and experimental evidence demonstrate non-cancer, cardiovascular, and endocrine effects of ionizing radiation exposure including growth hormone deficiency, obesity, metabolic syndrome, diabetes, and hyperinsulinemia. Insulin-like growth factor-1 (IGF-1) signaling perturbations are implicated in development of cardiovascular disease and metabolic syndrome. The minipig is an emerging model for studying radiation effects given its high analogy to human anatomy and physiology. Here we use a minipig model to study late health effects of radiation by exposing male Göttingen minipigs to 1.9-2.0 Gy X-rays (lower limb tibias spared). Animals were monitored for 120 days following irradiation and blood counts, body weight, heart rate, clinical chemistry parameters, and circulating biomarkers were assessed longitudinally. Collagen deposition, histolopathology, IGF-1 signaling, and mRNA sequencing were evaluated in tissues. Our findings indicate a single exposure induced histopathological changes, attenuated circulating IGF-1, and disrupted cardiac IGF-1 signaling. Electrolytes, lipid profiles, liver and kidney markers, and heart rate and rhythm were also affected. In the heart, collagen deposition was significantly increased and transforming growth factor beta-1 (TGF-beta-1) was induced following irradiation; collagen deposition and fibrosis were also observed in the kidney of irradiated animals. Our findings show Göttingen minipigs are a suitable large animal model to study long-term effects of radiation exposure and radiation-induced inhibition of IGF-1 signaling may play a role in development of late organ injuries.

Long-Term Changes in Cognition and Physiology after Low-Dose 16O Irradiation.

Astronauts traveling to Mars will be exposed to high levels of ionizing radiation upon leaving low-Earth orbit. During prolonged space travel, astronauts are exposed to galactic cosmic rays (GCRs) composed of protons; oxygen molecules; and high energy, high mass charged particles. Notably, oxygen molecules can travel through the shielding of spacecraft, potentially impacting 25% of the hippocampus. The aim of the current study was to assess whether 16O-particle radiation induced a behavioral deficit and histological changes in mice. Mice were sent to the National Aeronautics and Space Administration (NASA) Space Radiation Laboratory at Brookhaven National Laboratory and exposed to particulate 16O radiation at doses of 0 and 0.05 Gy. Nine months after irradiation, the mice were tested for novel object recognition and in the Y-maze, after which the animals were sacrificed. The brains were then dissected along the midsagittal plane for Golgi staining. Exposure to 0.05 Gy significantly impaired novel object recognition. However, short term memory and exploratory activity in the Y-maze were not affected. Micromorphometric analysis revealed significant decreases in mushroom spine density in the dentate gyrus and cornu Ammonis-1 and -3 of the hippocampus. Sholl analysis revealed a significant decrease in dendritic complexity in the dentate gyrus. The present data provide evidence that space radiation has deleterious effects on mature neurons associated with hippocampal learning and memory.

Impact of Spaceflight and Artificial Gravity on the Mouse Retina: Biochemical and Proteomic Analysis.

Astronauts are reported to have experienced some impairment in visual acuity during their mission on the International Space Station (ISS) and after they returned to Earth. There is emerging evidence that changes in vision may involve alterations in ocular structure and function. To investigate possible mechanisms, changes in protein expression profiles and oxidative stress-associated apoptosis were examined in mouse ocular tissue after spaceflight. Nine-week-old male C57BL/6 mice (n = 12) were launched from the Kennedy Space Center on a SpaceX rocket to the ISS for a 35-day mission. The animals were housed in the mouse Habitat Cage Unit (HCU) in the Japan Aerospace Exploration Agency (JAXA) "Kibo" facility on the ISS. The flight mice lived either under an ambient microgravity condition (µg) or in a centrifugal habitat unit that produced 1 g artificial gravity (µg + 1 g). Habitat control (HC) and vivarium control mice lived on Earth in HCUs or normal vivarium cages, respectively. Quantitative assessment of ocular tissue demonstrated that the µg group induced significant apoptosis in the retina vascular endothelial cells compared to all other groups (p < 0.05) that was 64% greater than that in the HC group. Proteomic analysis showed that many key pathways responsible for cell death, cell repair, inflammation, and metabolic stress were significantly altered in µg mice compared to HC animals. Additionally, there were more significant changes in regulated protein expression in the µg group relative to that in the µg + 1 g group. These data provide evidence that spaceflight induces retinal apoptosis of vascular endothelial cells and changes in retinal protein expression related to cellular structure, immune response and metabolic function, and that artificial gravity (AG) provides some protection against these changes. These retinal cellular responses may affect blood⁻retinal barrier (BRB) integrity, visual acuity, and impact the potential risk of developing late retinal degeneration.

The Vitamin E Analog Gamma-Tocotrienol (GT3) Suppresses Radiation-Induced Cytogenetic Damage.

PURPOSE: Ionizing radiation (IR) generates reactive oxygen species (ROS), which cause DNA double-strand breaks (DSBs) that are responsible for cytogenetic alterations. Because antioxidants are potent ROS scavengers, we determined whether the vitamin E isoform γ-tocotrienol (GT3), a radio-protective multifunctional dietary antioxidant, can suppress IR-induced cytogenetic damage. METHODS: We measured DSB formation in irradiated primary human umbilical vein endothelial cells (HUVECs) by quantifying the formation of γ-H2AX foci. Chromosomal aberrations (CAs) were analyzed in irradiated HUVECs and in the bone marrow cells of irradiated mice by conventional and fluorescence-based chromosome painting techniques. Gene expression was measured in HUVECs with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: GT3 pretreatment reduced DSB formation in HUVECS, and also decreased CAs in HUVECs and mouse bone marrow cells after irradiation. Moreover, GT3 increased expression of the DNA-repair gene RAD50 and attenuated radiation-induced RAD50 suppression. CONCLUSIONS: GT3 attenuates radiation-induced cytogenetic damage, possibly by affecting RAD50 expression. GT3 should be explored as a therapeutic to reduce the risk of developing genetic diseases after radiation exposure.

Synthesis of (2R,8' S,3' E)-δ-tocodienol, a tocoflexol family member designed to have a superior pharmacokinetic profile compared to δ-tocotrienol.

A group of side chain partially saturated tocotrienol analogues, namely tocoflexols, have been previously designed in an effort to improve the pharmacokinetic properties of tocotrienols. (2R,8'S,3'E,11'E)-δ-Tocodienol (1) was predicted to be a high value tocoflexol for further pharmacological evaluation. We now report here an efficient 8-step synthetic route to compound 1 utilizing naturally-occurring δ-tocotrienol as a starting material (24% total yield). The key step in the synthesis is oxidative olefin cleavage of δ-tocotrienol to afford the chroman core of 1 with retention of chirality at the C-2 stereocenter.

Molecular dynamics guided design of tocoflexol: a new radioprotectant tocotrienol with enhanced bioavailability.

There is a pressing need to develop safe and effective radioprotector/radiomitigator agents for use in accidental or terrorist-initiated radiological emergencies. Naturally occurring vitamin E family constituents, termed tocols, that include the tocotrienols, are known to have radiation-protection properties. These agents, which work through multiple mechanisms, are promising radioprotectant agents having minimal toxicity. Although α-tocopherol (AT) is the most commonly studied form of vitamin E, the tocotrienols are more potent than AT in providing radioprotection and radiomitigation. Unfortunately, despite their very significant radioprotectant activity, tocotrienols have very short plasma half-lives and require dosing at very high levels to achieve necessary therapeutic benefits. Thus, it would be highly desirable to develop new vitamin E analogues with improved pharmacokinetic properties, specifically increased elimination half-life and increased area under the plasma level versus time curve. The short elimination half-life of the tocotrienols is related to their low affinity for the α-tocopherol transfer protein (ATTP), the protein responsible for maintaining the plasma level of the tocols. Tocotrienols have less affinity for ATTP than does AT, and thus have a longer residence time in the liver, putting them at higher risk for metabolism and biliary excretion. We hypothesized that the low-binding affinity of tocotrienols to ATTP is due to the relatively more rigid tail structure of the tocotrienols in comparison with that of the tocopherols. Therefore, compounds with a more flexible tail would have better binding to ATTP and consequently would have longer elimination half-life and, consequently, an increased exposure to drug, as measured by area under the plasma drug level versus time curve (AUC). This represents an enhanced residence of drug in the systemic circulation. Based on this hypothesis, we developed a new class of vitamin E analogues, the tocoflexols, which maintain the superior bioactivity of the tocotrienols with the potential to achieve the longer half-life and larger AUC of the tocopherols.

A pooled analysis of nine studies in one institution to assess effects of whole heart irradiation in rat models.

PURPOSE: Over the years, animal models of local heart irradiation have provided insight into mechanisms of and treatments for radiation-induced heart disease in human populations. However, it is not completely clear which manifestations of radiation injury are most commonly seen after whole heart irradiation, and whether certain biological factors impact experimental results. Combining 9 homogeneous studies in rat models of whole heart irradiation from one laboratory, we sought to identify experimental and/or biological factors that impact heart outcomes. We evaluated the usefulness of including (1) heart rate and (2) bodyweight as covariates when analyzing biological parameters, and (3) we determined which echocardiography, histological, and immunohistochemistry parameters are most susceptible to radiation effects. Finally, (4) as an educational example, we illustrate a hypothetical sample size calculation for a study design commonly used in evaluating radiation modifiers, using the pooled estimates from the 9 rat studies only for context. The results may assist investigators in the design and analyses of pre-clinical studies of whole heart irradiation. MATERIALS AND METHODS: We made use of data from 9 rat studies from our labs, 8 published elsewhere in 2008-2017, and one unpublished study. Echocardiography, histological, and immunohistochemical parameters were collected from these studies. Using mixed effects analysis of covariance models, we estimated slopes for heart rate and bodyweight and estimated the radiation effect on each of the parameters. RESULTS: Bodyweight was related to most echocardiography parameters, and heart rate had an effect on echocardiography parameters related to the diameter of the left ventricle. For some parameters, there was evidence that heart rate and bodyweight relationships with the parameter depended on whether the rats were irradiated. Radiation effects were found in systolic measures of echocardiography parameters related to the diameter of the left ventricle, with ejection fraction and fractional shortening, with atrial wall thickness, and with histological measures of capillary density, collagen deposition, and mast cells infiltration in the heart. CONCLUSION: Accounting for bodyweight, as well as heart rate, in analyses of echocardiography parameters should reduce variability in estimated radiation effects. Several echocardiography and histological parameters were particularly susceptible to whole heart irradiation, showing robust effects compared to sham-irradiation. Lastly, we provide an example approach for a sample size calculation that will contribute to a rigorous study design and reproducibility in experiments studying radiation modifiers.

Genetic Upregulation of Activated Protein C Mitigates Delayed Effects of Acute Radiation Exposure in the Mouse Plasma.

Exposure to ionizing radiation, accidental or intentional, may lead to delayed effects of acute radiation exposure (DEARE) that manifest as injury to organ systems, including the kidney, heart, and brain. This study examines the role of activated protein C (APC), a known mitigator of radiation-induced early toxicity, in long-term plasma metabolite and lipid panels that may be associated with DEARE in APCHi mice. The APCHi mouse model used in the study was developed in a C57BL/6N background, expressing the D168F/N173K mouse analog of the hyper-activatable human D167F/D172K protein C variant. This modification enables increased circulating APC levels throughout the mouse's lifetime. Male and female cohorts of C57BL/6N wild-type and APCHi transgenic mice were exposed to 9.5 Gy γ-rays with their hind legs shielded to allow long-term survival that is necessary to monitor DEARE, and plasma was collected at 6 months for LC-MS-based metabolomics and lipidomics. We observed significant dyslipidemia, indicative of inflammatory phenotype, upon radiation exposure. Additionally, observance of several other metabolic dysregulations was suggestive of gut damage, perturbations in TriCarboxylic Acid (TCA) and urea cycles, and arginine metabolism. We also observed gender- and genotype-modulated metabolic perturbations post radiation exposure. The APCHi mice showed near-normal abundance for several lipids. Moreover, restoration of plasma levels of some metabolites, including amino acids, citric acid, and hypoxanthine, in APCHi mice is indicative of APC-mediated protection from radiation injuries. With the help of these findings, the role of APC in plasma molecular events after acute γ-radiation exposure in a gender-specific manner can be established for the first time.

AOP report: Development of an adverse outcome pathway for deposition of energy leading to abnormal vascular remodeling.

Cardiovascular diseases (CVDs) are complex, encompassing many types of heart pathophysiologies and associated etiologies. Radiotherapy studies have shown that fractionated radiation exposure at high doses (3-17 Gy) to the heart increases the incidence of CVD. However, the effects of low doses of radiation on the cardiovascular system or the effects from space travel, where radiation and microgravity are important contributors to damage, are not clearly understood. Herein, the adverse outcome pathway (AOP) framework was applied to develop an AOP to abnormal vascular remodeling from the deposition of energy. Following the creation of a preliminary pathway with the guidance of field experts and authoritative reviews, a scoping review was conducted that informed final key event (KE) selection and evaluation of the Bradford Hill criteria for the KE relationships (KERs). The AOP begins with a molecular initiating event of deposition of energy; ionization events increase oxidative stress, which when persistent concurrently causes the release of pro-inflammatory mediators, suppresses anti-inflammatory mechanisms and alters stress response signaling pathways. These KEs alter nitric oxide levels leading to endothelial dysfunction, and subsequent abnormal vascular remodeling (the adverse outcome). The work identifies evidence needed to strengthen understanding of the causal associations for the KERs, emphasizing where there are knowledge gaps and uncertainties in both qualitative and quantitative understanding. The AOP is anticipated to direct future research to better understand the effects of space on the human body and potentially develop countermeasures to better protect future space travelers.

Pharmacologic or genetic interference with atrogene signaling protects from glucocorticoid-induced musculoskeletal and cardiac disease.

Despite their beneficial actions as immunosuppressants, glucocorticoids (GC) have devastating effects on the musculoskeletal and cardiac systems, as long-term treated patients exhibit high incidence of falls, bone fractures, and cardiovascular events. Herein, we show that GC upregulate simultaneously in bone, skeletal muscle, and the heart, the expression of E3 ubiquitin ligases (atrogenes), known to stimulate the proteasomal degradation of proteins. Activation of Vitamin D receptor (VDR) signaling with the VDR ligands 1,25D3 (calcitriol, 1,25-dihydroxyvitamin D3) or ED (eldecalcitol, 2ß-(3-hydroxypropyloxy)-1,25-dihydroxyvitamin D3) prevented GC-induced atrogene upregulation in vivo and ex vivo in bone/muscle organ cultures and preserved tissue structure/mass and function of three tissues in vivo. Direct pharmacologic inhibition of the proteasome with carfilzomib also conferred musculoskeletal protection. Genetic loss of the atrogene MuRF1-mediated protein ubiquitination in ∆RING mice afforded temporary or sustained protection from GC excess in bone, or skeletal and heart muscle, respectively. We conclude that the atrogene pathway downstream of MuRF1 underlies GC action in bone, muscle, and the heart, and it can be pharmacologically or genetically targeted to confer protection against the damaging actions of GC simultaneously in the three tissues.

Cardiovascular effects after low-dose exposure and radiotherapy: what research is needed?

The authors of this report met at the Head Quarter of the International Atomic Energy Agency (IAEA) in Vienna, Austria, on 2-4 July 2012, for intensive discussions of an abundance of original publications on new epidemiological studies on cardiovascular effects after low-dose exposure and radiotherapy and radiobiological experiments as well as several comprehensive reviews that were published since the previous meeting by experts sponsored by the IAEA in June 2006. The data necessitated a re-evaluation of the situation with special emphasis on the consequences current experimental and clinical data may have for clinical oncology/radiotherapy and radiobiological research. The authors jointly arrived at the conclusions and recommendations presented here.

TLR4-A Pertinent Player in Radiation-Induced Heart Disease?

The heart is one of the organs that is sensitive to developing delayed adverse effects of ionizing radiation (IR) exposure. Radiation-induced heart disease (RIHD) occurs in cancer patients and cancer survivors, as a side effect of radiation therapy of the chest, with manifestation several years post-radiotherapy. Moreover, the continued threat of nuclear bombs or terrorist attacks puts deployed military service members at risk of exposure to total or partial body irradiation. Individuals who survive acute injury from IR will experience delayed adverse effects that include fibrosis and chronic dysfunction of organ systems such as the heart within months to years after radiation exposure. Toll-like receptor 4 (TLR4) is an innate immune receptor that is implicated in several cardiovascular diseases. Studies in preclinical models have established the role of TLR4 as a driver of inflammation and associated cardiac fibrosis and dysfunction using transgenic models. This review explores the relevance of the TLR4 signaling pathway in radiation-induced inflammation and oxidative stress in acute as well as late effects on the heart tissue and the potential for the development of TLR4 inhibitors as a therapeutic target to treat or alleviate RIHD.

Effects of Whole and Partial Heart Irradiation on Collagen, Mast Cells, and Toll-like Receptor 4 in the Mouse Heart.

In radiation therapy of tumors in the chest, such as in lung or esophageal cancer, part of the heart may be situated in the radiation field. This can lead to the development of radiation-induced heart disease. The mechanisms by which radiation causes long-term injury to the heart are not fully understood, but investigations in pre-clinical research models can contribute to mechanistic insights. Recent developments in X-ray technology have enabled partial heart irradiation in mouse models. In this study, adult male and female C57BL/6J mice were exposed to whole heart (a single dose of 8 or 16 Gy) and partial heart irradiation (16 Gy to 40% of the heart). Plasma samples were collected at 5 days and 2 weeks after the irradiation for metabolomics analysis, and the cardiac collagen deposition, mast cell numbers, and left ventricular expression of Toll-like receptor 4 (TLR4) were examined in the irradiated and unirradiated parts of the heart at 6 months after the irradiation. Small differences were found in the plasma metabolite profiles between the groups. However, the collagen deposition did not differ between the irradiated and unirradiated parts of the heart, and radiation did not upregulate the mast cell numbers in either part of the heart. Lastly, an increase in the expression of TLR4 was seen only after a single dose of 8 Gy to the whole heart. These results suggest that adverse tissue remodeling was not different between the irradiated and unirradiated portions of the mouse heart. While there were no clear differences between male and female animals, additional work in larger cohorts may be required to confirm this result, and to test the inhibition of TLR4 as an intervention strategy in radiation-induced heart disease.

Effects of Simulated 5-Ion Galactic Cosmic Radiation on Function and Structure of the Mouse Heart.

Missions into deep space will expose astronauts to the harsh space environment, and the degenerative tissue effects of space radiation are largely unknown. To assess the risks, in this study, male BALB/c mice were exposed to 500 mGy 5-ion simulated GCR (GCRsim) at the NASA Space Radiation Laboratory. In addition, male and female CD1 mice were exposed to GCRsim and administered a diet containing Transforming Growth Factor-beta (TGF-ß)RI kinase (ALK5) inhibitor IPW-5371 as a potential countermeasure. An ultrasound was performed to investigate cardiac function. Cardiac tissue was collected to determine collagen deposition, the density of the capillary network, and the expression of the immune mediator toll-like receptor 4 (TLR4) and immune cell markers CD2, CD4, and CD45. In male BALB/c mice, the only significant effects of GCRsim were an increase in the CD2 and TLR4 markers. In male CD1 mice, GCRsim caused a significant increase in total collagens and a decrease in the expression of TLR4, both of which were mitigated by the TGF-ß inhibitor diet. In female CD1 mice, GCRsim caused an increase in the number of capillaries per tissue area in the ventricles, which may be explained by the decrease in the left ventricular mass. However, this increase was not mitigated by TGF-ß inhibition. In both male and female CD1 mice, the combination of GCRsim and TGF-ß inhibition caused changes in left ventricular immune cell markers that were not seen with GCRsim alone. These data suggest that GCRsim results in minor changes to cardiac tissue in both an inbred and outbred mouse strain. While there were few GCRsim effects to be mitigated, results from the combination of GCRsim and the TGF-ß inhibitor do point to a role for TGF-ß in maintaining markers of immune cells in the heart after exposure to GCR.